CHARACTERIZATION OF AN ACTIN-DEPENDENT ORGANELLE MOTOR

肌动蛋白依赖性细胞器运动的表征

• 批准号: 2185900
• 负责人: George M. Langford
• 金额: $ 3.1万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185900
• 关键词: Cephalopoda; actins; alternatives to animals in research; axon; calmodulin; electron microscopy; freeze etching; intracellular transport; microfilaments; neuronal transport; organelles; phosphorylation; protein reconstitution; protein structure; saltwater environment

DESCRIPTION (Adapted from the applicant's abstract): Organelle movement in higher animal cells, especially in neurons, is known to be microtubule- based. Using axoplasm from squid giant axons the investigators have found that organelles exhibit, in addition to the well known microtubule- dependent motility, an equally prominent novel type of motility associated with an extensive actin filament network. This actin-associated movement is ATP-dependent, has myosin-like pharmacology, is unidirectional, and is independent of microtubules and microtubule-based motors. These findings show that actin-based organelle motility is driven by a myosin like motor and represent a major component of fast axonal organelle transport. Actin-based motility appears to operate in close association with microtubule-dependent motility to produce and regulate the movement of organelles. The goal of this proposal is to isolate this motor from squid giant axons and optic lobes and characterize its structure, function and regulation. The specific aims are as follows. The investigator will identify and isolate the myosin-like motor by subcellular fractionation of neural tissues from aquid (axoplasm and optic lobes). Experiments will be performed to obtain a purified preparation of active motor protein. After purification of the motor, the investigators will determine its molecular structure, enzymatic properties and ability to move organelles along actin filaments. Using an in vitro reconstituted system for actin-based organelle motility, the investigators will determine the mechanism by which the functional activities of the myosin-like motors are regulated. The investigators propose that calmodulin, via phosphorylation- dephosphorylation reactions, is involved in its regulation. The investigators will study the fine structure of the myosin-like motors and the organelle-motor-microfilament complexes using platinum shadowing, negative contrast and freeze-fracture electron microscopy.

描述（改编自申请人的摘要）：Organelle运动 在较高的动物细胞中，尤其是在神经元中，已知是微管 基于。使用鱿鱼巨型轴突的轴突发现 除了众所周知的微管外，细胞器还表现出 依赖运动性，一种同样突出的新型运动类型 具有广泛的肌动蛋白细丝网络。 这种与肌动蛋白相关的运动 是依赖ATP的，具有肌球蛋白样药理学，是单向的，是 独立于微管和基于微管的电动机。 这些发现 表明基于肌动蛋白的细胞器运动是由像电机这样的肌球蛋白驱动的 并代表快速轴突细胞器转运的主要组成部分。 基于肌动蛋白的运动似乎与 微管依赖性运动能够产生和调节 细胞器。 该提议的目的是将此电动机与鱿鱼隔离开来 巨型轴突和视线叶，并表征其结构，功能和 规定。 具体目标如下。调查员会 通过亚细胞分级分馏，识别和隔离肌球蛋白样电机 来自水平的神经组织（轴质和视神经裂片）。 实验将是 进行以获得活性运动蛋白的纯化制备。 后 电机的纯化，研究人员将确定其分子 结构，酶特性和沿肌动蛋白移动细胞器的能力 细丝。 使用基于肌动蛋白的体外重构系统 细胞器运动性，研究人员将通过 肌球蛋白样电机的功能活动受到调节。 研究人员建议通过磷酸化 - 钙调蛋白 - 去磷酸化反应与其调节有关。 这 研究人员将研究类似肌球蛋白的电动机的精细结构和 使用白金阴影的细胞器 - 机动微丝复合物， 负鲜明对比和冻结电子显微镜。